Reviews on Using NMR and Other Method on Analyzing Algae Lipid
Qian Zhou, Shulin Chen
(BBEL, Washington State University, Pullman, WA, 99164)
Abstract:
This paper is a review on evaluation of using NMR as a tool to analyze algae lipid for
biofuels production. Therefore, we compared several commonly used NMR with other
traditional methods of analyzing algae lipid. Both of the advantage and disadvantage of
those methods are compared separately. Although there is no quantitative method to
determine the algae lipid by ordinary NMR, but a qualitative analysis of lipid content in
green algae has become reality. So, we can predict that in the near future, quantitative
analysis of algae lipid by ordinary NMR should be possible. These results indicate that
NMR is a useful diagnostic tool for screening high lipids algae and identifying lipids in
algae, with particular relevance to biofuel production.
Key words: NMR, algae, lipid, biofuel, biodiesel
Introduction
Due to the limited stocks of fossil fuels and the increasing emission of greenhouse gas
carbon dioxide into the atmosphere from the combustion of fossil fuels, research has
begun to focus on alternative biomass-derived fuels. Biodiesel produced from algae is a
worldwide studied potential alternative to fossil fuels based transportation fuels. The need
to find substitution of traditional fossil fuel has been attracting an increasingly more
attention in nowadays.
The use of microalgae to produce lipid from either phothtrophical or heterotrophical way
has gain the numerous researchers’ vision.
The need to replace fossil fuels with carbon-neutral and renewable biofuels has led to a
tremendous amount of research in determining which biologically based systems are
going to take a lead role in this enormous undertaking. The use of micro-algae to produce
a lipid compliment either phototrophically or heterotrophically has gained a lot of
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attentions. Micro-algae have many advantages over terrestrial plants as a possible source
of high quality lipids in sufficient quantities as to be competitive with fossil fuels as the
price of the latter continue to rise.
In the production of these lipids it is imperative that methods be found that can easily
quantify the lipid fraction as the organism begins to produce it. Ideally the methods
would be able to monitor the production of lipids within the culture throughout the entire
growth cycle, be accurate with respect to total lipid content, be able to identify the
particular lipids being produced and be able to do the measurements without extensive
handling or manipulation of the growing cultures. In addition the method should be able
to deliver feedback to the production personnel as to how changes in the culture
conditions are reflected in the lipid content as well as giving a real-time determination of
when to harvest the mature cells.
The conventional method used for lipid determination involves solvent extraction and
gravimetric determination (Bligh and Dyer, 1959). Further quantification of neutral lipids
requires the separation of the crude extractions and quantification of the lipid fractions by
thin-layer chromatography (TLC), HPLC or gas chromatography (GC) (Eltgroth et al.,
2005).
Gravity method
Total oil content is generally estimated by Soxhlet (AOAC, 1980) or by cold extraction
methods (Bligh and Dyer, 1959; Folch et al., 1957) using organic solvents and the fatty
acid composition of the extracted oil is determined by gas liquid chromatography after
trans-methylation. Oil extraction followed by esterification is cumbersome, time
consuming, and often uneconomical.
GC-MS
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The current state of lipid analysis requires a variety of post harvest steps that severely
hinder the ability to make quick decisions about the culture. The lipid content is most
often measured by taking an aliquot of the culture, centrifuging the cells into a pellet,
drying the pellet by lyophilization, disruption of the cells followed by exhaustive solvent
extraction. The lipid content is then weighed after the solvent has been removed and the
percent lipid determined from the weight of the lyophilized cells. To obtain the chemical
identity of the lipids the extracted lipids are subsequently saponified with strong base
followed by methyl esterification and GC-MS analysis.
Nile Red Staining
In recent decades, Nile Red (NR) staining seems preferable in fluorescent measurement
of intracellular lipid because the method is quick and simple. NR, a red phenoxazine dye,
serves as a lipid probe due to its specific interaction with hydrophobic molecules (Fowler
and Greenspan, 1985). Again this method suffers from the need for several post-harvest
manipulations and the inability to accurately quantify low lipid content levels. Recently a
method has been published that comes close to meeting all of the criteria for a robust and
rapid analysis method.
Recently, Chen et al. (2009) use Nile red staining to measure algae neutral lipids in high
throughput way.
But, the cell wall differs in various algae so that Nile may be applied well in several kinds
of algae but does not work in others, especially when the cell wall is thick and rigid the
Nile red cannot penetrate into the cell of the algae.
New Method Coming--NMR
A major drawback of the conventional method is that it is time- and labor-intensive,
making it difficult to screen large numbers of algae.
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TD NMR
The solids such as carbohydrate and protein exhibit the shortest relaxation times in the
order of microseconds, whereas the relaxation times for bound water, free water and lipid
are a few hundreds of microseconds, seconds and a few hundreds of milliseconds,
respectively (Todt et al., 2006; Kenar, 2007).The TD-NMR measurements involve very
little sample preparation, except for placing the sample inside the probe. No chemical
change occurs to the sample as it undergoes the measurement. Most of the measurements
can be accomplished in seconds. One instrument can be used to analyze multiple physicochemical properties through use of proper pulse-sequences. The whole measurement
process can be completely automated using available robotic accessories. For example,
the % H content measurement in hydrocarbon samples involved filling up the 18 mm
diameter tubes up to a height of ~3 cm and loading them to the autosampler rack, in terms
of sample preparation. The whole measurement sequence was automated and took 8 s for
each sample.
Compared with ordinary NMR, TD-NMR is faster, easier to use and less expensive. TDNMR is based on the different relaxation times of hydrogen nuclei in different phases of
the sample analyzed (Todt et al., 2001). The solids such as carbohydrate and protein
exhibit the shortest relaxation times in the order of microseconds, whereas the relaxation
times for bound water, free water and lipid are a few hundreds of microseconds, seconds
and a few hundreds of milliseconds, respectively (Todt et al., 2006; Kenar, s2007).
Recently Gao et al (2008) published a method using time-domain NMR (TD-NMR) to
analyze the mobile lipid content within lyophilized algal cells. Whereas the TD-NMR
method proved to be highly accurate (even at low lipid concentration) when compared
with the extractive-gravimetric methods and the Nile Red staining methods the need to
centrifuge and lyophilize the cells removes it from being able to determine lipid
concentrations in real time.
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HR-MAS NMR
Highresolution magic angle spinning (HR MAS), where samples are spun at low
frequency around their own axes at an angle of 54.7° relative to the magnetic field, has
the effect on semi-solid or solid samples that it decreases spectral line broadening due to
molecular anisotropic interactions (Andrew 1981).
Analysis of metabolites (including lipids) within viable algal cells and oil-seeds has also
been done using what is known as high-resolution magic angle spinning NMR (HRMAS). HR-NMR spectra of cells results in high resolution 1H and 13C spectra of mobile
metabolites which are close to the resolution obtainable in traditional solution-state NMR.
Examples of the use of HR-MAS to investigate algal metabolism are Bondu et al who
investigate carbohydrates within re-algal (Rhodophyta), Broberg et al who investigated
low molecular weight metabolites in other red algal species and Chaunton et al who used
HR-MAS to perform metabolomic studies on a variety of omarine green algae and
diatom species. Lastly Hutton et al used HR-MAS to investigate the lipid content and
lipid identity within canola oil seeds.
The use of HR-MAS as an analysis tool comes very close to meeting all of the criteria for
rapid and robust method for determination of the content and identity of lipids within
growing algal cultures. The only limitation on its sue comes from the need to centrifuge
the cell culture and in most instances reported in the literature the cells are then washed
with D2O as a way of removing most of the exogenous H2O as well as provide a lock
signal. Addition of an external standard (TSP or DSS) of known concentration allows the
researcher to compare directly the integrated intensity of the lipid envelope with that of
the known standard. Alternatively the ERETIC method of electronic reference signal
injection can be used. If the corresponding wet weight of the cells and subsequently the
dry weight are know then a rapid determination of oil content can be derived. As a bonus
the identity of the lipids can also be derived via 2D HSQC spectra (20 minutes) and
comparison with 2D HSQC of standards tri-acyl glycerides. Whereas this has not yet
been reported in the literature (as far as we know) it can be demonstrated with synthetic
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mixtures.
Liquid State NMR
Currently, NMR on small liquid samples at room temperature is performed suing small
solenoids wrapped around a capillary or planar coils on glass chips containing
microfluidic channels. However, this approache suffer from spectral resolution problems,
mainly induced by the nearby windings of the microcoil that distort the static magnetic
field. To overcome this problem, Jacob etc. introduced a new NMR coil, they using
tripline instead.
Lastly the use of standard high resolution liquid-state NMR, either in a standard tube
geometry or better in a flow-cell equipped probe has not been reported used for
determination of oil content in algal cells. With this method there is no centrifugation step
and in the case of flow-cell equipped probes the culture can drawn directly from the bioreactor into the NMR probe for analysis. To investigate the efficacy of using standard
liquid-state NMR for analysis of lipids in whole algal cells we took 0.7 ml of algal
culture, added 50ul of D2O and placed the suspension into a standard 5mm NMR tube.
The sample was analyzed on a Varian VNMRS 600 MHz spectrometer using a standard
presaturation pulse sequence. The water peak was suppressed using a 1.5s presaturation
pulse at 6dB.
The most visible feature of the algal spectrum is that the lines a quite broad compared to
those in the spectrum of the oleic TAG. The spectrum of the lipid content inside the cell is
possible due to the fact that lipid within the lipid vesicles has a fast correlation time
compared to that of the more rigid cellular components. The line broadening in the
spectrum is mainly a result of the difference in magnetic susceptibility between the algal
cells and the surrounding aqueous environment. It is this magnetic susceptibility
broadening that is removed via spinning at the magic angle in HR-MAS spectroscopy.
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We propose the use of flow cell probe in combination with HR-MAS to perform rapid
lipid analysis of growing algal cultures. In the first instance the overall lipid
concentration can be evaluated using the flow cell probe by allowing a sample of the
dispersed algal culture to be drawn up into the flow cell and measured via presaturation
1H spectroscopy. Ideally the sample would be drawn continuously into the probe at a
flow rate whereby the sampling rate could be matched with the time it takes for the algae
at the beginning of a scan to exit by the time data acquisition has finished for that scan. In
this fashion the scan rate can adjusted to be fairly rapid while still having the incoming
nuclear spins to be a full Boltzman distribution. In addition there would be no settling of
the cells during a flow measurement. An internal standard such as TSP or DSS could be
used to enable quantization or better yet the ERETIC method could be used elimination
the need to introduce exogenous materal.
The actual identity of the lipid would be done with parallel samples that would be
analyzed using HR-MAS. Since the samples would be centrifuged a wet weight per ml of
culture media could determined. The HR-MAS studies would be longer term and only
performed when the specific lipid identity is needed. HR-MAS 1H and 1H detected 2D 1H
13
C HSQC spectra would be obtained and analyzed for lipid identity and mole fraction.
Beal et al (2010) has discovered a qualitative way to analyze green alga lipid by liquid
state NMR
Future Use of NMR on algae
NMR provides information that is not available from other analysis tools, such as specific
chemical structure, and can be used to analyze bulk samples of intact algae. Thus,
ordinary liquid state NMR can be a useful complementary analysis tool to supplement
data provided by other analysis methods (e.g., solvent extraction and chromatography).
The former study demonstrates the probability and principle for the use NMR to identify
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the presence of triglyceride in algal samples. Triglyceride was determined to be present in
the algal cultures by comparing the 13C NMR algal spectra to that of a pure triglyceride
standard (glyceryl trioleate). Every peak in the standard triglyceride spectrum was also
present in the algal spectra (healthy and nitrogen-starved). In addition, the nitrogenstarved culture produced methylene signals (29.5 ppm) that were, on average, 2.5 times
greater than those of the healthy culture. This result was expected because nitrogenstarved algae contain greater lipid content and further supports the ability of NMR to
evaluate algal lipid content. Interference analysis indicated that mono- and diglycerides
were also likely present in the algae, and that these compounds were more prevalent in
the healthy culture than the starved culture. Characteristic phospholipid signals were not
distinct in the 13C NMR algal spectra (likely due to inhomogeneous field line broadening)
but were present in 31P NMR spectra. Thus, it is probable that the methylene peaks in the
algal spectra contain contributions from mono-, di-, and triglycerides and may possibly
contain contributions fromphospholipids. The extent of each contribution is not known.
Therefore, the methylene peaks in the 13C NMR spectra indicate the content of a broad
range of lipids, while compound-specific peaks are needed to identify particular
compounds, such as the characteristic triglyceride peaks at 62.1 and 68.9 ppm. While this
exploratory study verifies the potential viability of NMR as a diagnostic tool, the
sensitivity or quantitative capability of ordinary NMR for algal lipids was not
investigated. Because this proof-of-principle study demonstrates the application of 13C
NMR analysis for one alga that was grown in the laboratory, further research is
recommended to determine the ability for NMR to characterize lipids in a range of algal
species and cultures grown under different conditions. Furthermore, development of
postprocessing methods that improve the specificity of NMR to individual compounds of
interest would also be valuable. In conclusion, 13C NMR is a tool with potential for
analyzing intermediate products in the algal biodiesel production pathway, including
algal cultures.
Acknowledgement:
We would like to thank Greg Helms, William Hiscox, Ben Lucker and Zhanyou for their
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help and assistance during writing this review this paper.
This paper is supported by XXXXXXX funding from XXXXX and China Scholarship
Council (CSC).
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